Enhancement of osteogenic potential of bone grafts

ABSTRACT

The present invention concerns the enhancement of the osteogenic potential of bone graft by ex vivo treatment with a Wnt polypeptide, such as a liposomal Wnt polypeptide.

CROSS REFERENCE

This application claims benefit and is a Continuation of application ofSer. No. 14/333,220 filed Jul. 16, 2014, now U.S. Pat. No. 9,301,980,which claims benefit of U.S. Provisional Patent Application No.61/957,946, filed Jul. 16, 2013, which applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention concerns the enhancement of the osteogenicpotential of bone graft by ex vivo treatment with a Wnt polypeptide,such as a liposomal Wnt polypeptide. In particular, the inventionconcerns the ex vivo treatment of bone grafts with a Wnt3a protein,preferably liposomal Wnt3a (L-Wnt3a).

BACKGROUND OF THE INVENTION

Orthopedic and dental implants are used for a variety of joint and teethreplacements and to promote bone repair in humans and animals,particularly for hip and knee joint and tooth replacements. Althoughmany individuals experience uncomplicated healing and restoration offunction, there is also a high rate of complications, estimated at10-20% for total joint replacements. The majority of these failures andsubsequent revision surgeries are made necessary by failure at theimplant-bone interface. In addition, implants used as anchorage devicesfor orthodontic tooth movement have an estimated 40% failure rate andsubsequent placement of additional implants is made necessary because offailures at the implant-bone interface.

Orthopedic and dental implants are made of materials which arerelatively inert (“alloplastic” materials), typically a combination ofmetallic and ceramic or plastic materials. Previous approaches toimprove the outcomes of orthopedic implant surgeries have mainly focusedon physical changes to the implant surface designed to increased boneformation. These approaches include using implants with porous metallicsurfaces to promote bone ingrowth and spraying implants withhydroxyapatite plasma. Approaches using dental implants have alsoincluded the use of topographically-enhanced titanium surfaces in whichsurface roughness is imparted by a method such as grit blasting, acidetching, or oxidation.

Also in an effort to promote osseointegration, implant surfaces haveundergone major alterations. For example, short peptides containing anarginineglycineaspartic acid (RGD) sequences have been attached toimplant surfaces because cells utilize RGD sequences to attach to theextracellular matrix. Investigators have attempted to recreate this cellattachment to the modified implant surface but this strategy hasresulted in only modest increases in implant osseointegration andmechanical fixation. Alternatively, in an attempt to stimulate bloodvessel ingrowth around implants their surfaces have been coated with acoating containing the angiogenic growth factor VEGF. Implants soaked insaline solutions have been marketed as a means to increase implantosseointegration, with little or no data to substantiate the claims.

Another strategy employed to stimulate osseointegration is tonano-texture the implant surface. The rationale behind this strategy isthat texturing increases surface area and therefore prevents the implantfrom “sliding” against cells in the pen-implant environment. In clinicaltrials, however, nano-texturing does not result in measureable benefits.

The use of protein-based approaches to stimulate implantosseointegration has also been under intense investigation. RecombinantBone Morphogenetic Proteins (BMPs) induce robust bone formation inskeletal fractures and they have also been employed in an effort tostimulate direct bone formation around implants. While in vitro resultshave been encouraging, in vivo data are less convincing. RecombinantBMPs inhibit osteogenic differentiation of cells in the bone marrowcavity and consequently, are contraindicated for implantosseointegration. See Sykaras et al. (2004) Clin Oral Investig 8(4):196-205; and Minear et al. (2010) Journal of Bone and Mineral Research25(6): 1196-207. The use of BMPs has been associated with increasedincidence of heterotopic ossifications and uncontrolled inflammation andmore recent metadata analyses demonstrate an increased risk of cancersas well.

Wnt proteins form a family of highly conserved secreted signalingmolecules that bind to cell surface receptors encoded by the Frizzledand low-density lipoprotein receptor related proteins (LRPs). The WNTgene family consists of structurally related genes which encode secretedsignaling proteins. These proteins have been implicated in oncogenesisand in several developmental processes, including regulation of cellfate and patterning during embryogenesis. Once bound, the ligandsinitiate a cascade of intracellular events that eventually lead to thetranscription of target genes through the nuclear activity of β-cateninand the DNA binding protein TCF (Clevers H, 2004 Wnt signaling:lg-norrin the dogma. Curr Biol 14: R436-R437; Nelson W J, Nusse R 2004Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303:1483-1487; Gordon M D, Nusse 2006 Wnt signaling: Multiple pathways,multiple receptors, and multiple transcription factors. J Biol Chem 281:22429-22433).

Wnts are also involved in a wide variety of cellular decisionsassociated with the program of osteogenesis. For example, Wnts regulatethe expression levels of sox9 and runx2, which influences the commitmentof mesenchymal progenitor cells to a chondrogenic or an osteogenic cellfate. Wnts influence the rate of differentiation of osteoprogenitorcells. In adult animals there is abundant evidence that Wnt signalingregulates bone mass. For example, gain-of-function mutations in thehuman Wnt co-receptor LRP5 are associated with several high bone masssyndromes, including osteopetrosis type I, and endosteal hyperostosis orautosomal dominant osteosclerosis. Loss-of-Wnt-function mutations causelow bone mass diseases including osteoporosis-pseudoglioma. Increasedproduction of the Wnt inhibitor Dkk1 is associated with multiplemyeloma, a disease that has increased bone resorption as one of itsdistinguishing features. For further details, see, S. Minear et al., Wntproteins promote bone regeneration. Sci. Transl. Med. 2, 29ra30 (2010);Zhao et al., Controlling the in vivo activity of Wnt liposomes, MethodsEnzymol 465: 331-47 (2009); Popelut et al., The acceleration of implantosseointegration by liposomal Wnt3a, Biomaterials 31 9173e9181 (2010);and Morrell N T, Leucht P, Zhao L, Kim J-B, ten Berge D, et al. (2008)Liposomal Packaging Generates Wnt Protein with In Vivo BiologicalActivity. PLoS ONE 3(8): e2930.

It has been shown that combining Wnt proteins with lipid vesicles(liposomes) produced a Wnt formulation (Morrell et al., 2008, supra; andZhao et al., 2009, supra) with biological activity (Minear et al., 2010,supra; and Popelut et al., 2010, supra). The biological activity ofsoluble wingless protein is described in van Leeuwen et al. (1994)Nature 24: 368(6469): 3424. Biochemical characterization of Wnt-Frizzledinteractions using a soluble, biologically active vertebrate Wnt proteinis described by Hsieh et al. (1999) Proc Natl Acad Sci USA 96(7):3546-51. Bradley et al. (1995) Mol Cell Bioi 15(8): 4616-22 describe asoluble form of Wnt protein with mitogenic activity. The use ofliposomal Wnt proteins to enhance osseointegration is described in U.S.Patent Publication No. 20120115788.

SUMMARY OF THE INVENTION

In one aspect, the invention concerns a method of enhancing cellsurvival in a bone graft, comprising subjecting the bone graft to exvivo treatment with a Wnt polypeptide. In another aspect, the inventionconcerns a method of enhancing the osteogenic potential of a bone graft,comprising subjecting the bone graft to ex vivo treatment with aneffective dose of a Wnt polypeptide, including without limitation Wnt3A.In a further aspect, the invention concerns a method for revitalizing abone graft from a subject with diminished healing potential, comprisingsubjecting the bone graft to ex vivo treatment with a Wnt polypeptide.In all aspects, the bone graft may be an autograft or an allograft. Inall aspects, the bone graft may comprise a stem cell population, suchas, for example, a bone-marrow-derived stem cell population, e.g. bonemarrow-derived mesenchymal stem cells.

The bone graft preferably is from a human subject. In one embodiment,the human subject is an elderly patient. In certain embodiments, thehuman subject is at least 50 years old, at least 55 years old, at least60 years old, or at least 65 years old, or at least 70 years old, or atleast 75 years old, or at least 80 years old, or at least 85 years old.In another embodiment, the human subject has diminished healingpotential, e.g. is a smoker, diabetic, or a person characterized bynutritional deficits.

In all aspects and embodiments, the Wnt polypeptide preferably is Wnt3a,more preferably human Wnt3a, most preferably liposomal human Wnt3a(L-Wnt3a). In a further aspect, the methods of the present inventionfurther comprise the step of introducing the bone graft into a recipientsubject, such as a human patient. In various embodiments, the bone graftmay be used to support a dental implant, to repair a bone fracture. Inanother embodiment, the bone graft is used to repair or rebuild adiseased bone. In yet another embodiment, the bone graft is used in therecipient's hips, knees or spine.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Bone grafts have osteogenic potential. (A) Quantification oftotal DNA in representative aliquots of whole bone marrow harvested fromtransgenic beta actin-enhanced green fluorescent protein (β-actin-eGFP)male mice; each aliquot constitutes a bone graft. (B) Bone grafts aretransplanted into 2-mm diameter critical-size calvarial defects(demarcated with a circle), which are created in the sagittal suturethat separates the parietal bones (outlined with vertical white dashedlines). The dashed black line indicates the plane of tissue section. (C)Representative tissue section from the injury site on post transplantday 1; GFP immunostaining identifies grafted cells from the eGFP donor(n=5); the inferior space represents the sagittal sinus. (D)Representative tissue section on post-transplant day 5; immunostainingfor bromodeoxyuridine (BrdU) identifies cells in S phase. (E) On posttransplant day 7, GFP immunostaining identifies the bone graft (dottedyellow line); a higher magnification image of the boxed area in (E) (F)illustrates that the majority of the cells in the injury site arederived from GFP-positive graft. (G) On post-transplant day 14, micro-CTreconstruction confirms that a 2-mm calvarial injury constitutes acritical-size nonhealing defect (n=6) 40. (H) The same size calvarialinjury, treated with a bone graft, heals (n=6). (I) and (J) Onpost-transplant day 7, aniline blue staining was used to identify newosteoid matrix; no osteoid matrix formed in the untreated injury site(yellow dotted line). (J) shows visible osteoid matrix onpost-transplant day 7 in a representative sample that had been treatedwith a bone graft. Abbreviations: IHC=immunohistochemistry. Arrows markthe edges of intact bone. Scale bars: 2 mm (B); 200 μm (C) and (D) 100μm (E); 40 μm (F); 2 mm (G); and 200 μm (I) and (J).

FIG. 2. Osteogenic potential is reduced in bone grafts from agedanimals. On post-transplant day 7 (d7), aniline blue staining indicatesosteoid matrix generated by bone grafts from young (A) versus ageddonors (B). (C) Histomorphometric analyses of the amount of new boneformed from young and aged bone grafts. (D) On post-transplant day 7(d7), green fluorescent protein (GFP) immunostaining identifies cellsderived from the bone graft when the donor is young as compared withaged donors. (F) The number of GFP-positive (GFP^(+ve)) cells in theinjury site when the graft is harvested from young (blue bars, n=13)compared with aged (white bars, n=13) donors. On post-transplant day 5(d5), bromodeoxyuridine (BrdU) staining identifies proliferating cellsin bone grafts from young (G) and aged (H) donors. (I) Quantitativereverse transcription-polymerase chain reaction (qRTPCR) forproliferating cell nuclear antigen (PCNA) in bone grafts from young andaged animals are equivalent. Single asterisk denotes p<0.05. Arrowmarksthe edge of intact bone. Scale bars: 200 μm ((A), [scale bar in (A) alsoapplies to (B)], (D) [scale bar in (D) also applies to FIG. (E)], and(G) [scale bar in FIG. (G) also applies to (H)]).

FIG. 3. Wnt signaling is reduced in aged bone grafts. (A) QuantitativeRT-PCR to evaluate relative expression levels of Wnt ligands and Wnttarget (B) genes in bone marrow (BM) harvested from young (blue bars;n=3) and aged (white bars; n=3) donors. Gene expression levelsnormalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Asteriskdenotes p<0.05.

FIG. 4. Liposomal Wnt3a restores osteogenic capacity to aged bonegrafts. (A) Aniline blue staining of L-PBS treated aged bone grafts(n=5). (B) New aniline-blue positive osteoid matrix in L-Wnt3a treatedbone grafts (n=8). (C) Histomorphometric quantification of new bonematrix on post-transplant days seven and twelve. (D) Aniline bluestaining on post-transplant day twelve (d12) in L-PBS and L-Wnt3a (E)treated bone grafts. (F) Beta galactosidase (□-gal) activity normalizedto total DNA as measured in cell populations (unattached, floating cellsand attached cells) from a bone marrow harvest. White bars (n=4)represent Wnt responsiveness following L-PBS treatment; blue bars (n=4)represent Wnt responsiveness following L-Wnt3a treatment (effectiveconcentration 0.15 μg/mL Wnt3a). (G) Immunostaining for the stem cellmarkers CD45, CD73, CD105, and Strol in attached cells derived from thebone marrow. (H) Beta galactosidase activity normalized to total DNA inthe attached cell population following L-PBS treatment (white bars, n=4)or following L-Wnt3a treatment (n=4; effective concentration 0.15 μg/mLWnt3a). (I) Xgal staining on a representative tissue section identifiesWnt responsive cells in a bone graft from an aged Axin2^(LacZ/+) mousetreated with L-PBS, compared with treatment with L-Wnt3a (J). (K) Xgalstaining on a representative tissue section identifies Wnt responsivecells in an L-PBS-treated bone graft from a young Axin2^(LacZ/+) mouse.Single asterisk denotes p<0.05; quadruple asterisk denotes p<0.0001.Abbreviations: L-PBS=liposomal PBS; L-Wnt3a=liposomal Wnt3a; BM=bonemarrow; and DAPI=4′,6-diamidino-2-phenylindole, dihydrochloride. Arrowsmark the edges of intact bone. Scale bars: 100 mm ((A) [scale bar in (A)also applies to (B)]); 200 mm ((D) [scale bar in (D) also applies to(E)]); 100 mm (G); and 40 mm ((I), [scale bar in (I) also applies to (J)and (K)]).

FIG. 5. L-Wnt3a treatment restores osteogenic potential to bone graftsfrom aged animals. Bone marrow from aged donor rabbits, assayed for DNAfragmentation associated with cell apoptosis. (A) Terminaldeoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining(n=4) demonstrates the extent of apoptosis in aged bone marrow treatedwith L-PBS (10 mL), compared with L-Wnt3a (B) treatment (effectiveconcentration=0.15 μg/mL Wnt3a). (C) A measurement of caspase activityin aged bone graft samples treated with L-PBS (white bars) or L-Wnt3a(blue bars). (D) through (G) Bone marrow was harvested from agedrabbits, incubated with L-PBS or L-Wnt3a for up to 1 h, thentransplanted into a critical-size defect created in the ulna. (D)Radiographic assessment at four weeks following bone-grafting. CompareL-PBS treatment with L-Wnt3a (E) treatment. (F) Micro-CT iso-surfacereconstruction at eight weeks following bone-grafting. Compare L-PBStreatment with L-Wnt3a (G) treatment. (H) Bone volume (BV) and bonevolume/total volume (BV/TV) are calculated using the bone analysis toolin GE MicroView software. A single asterisk denotes p<0.05.Abbreviations: L-PBS=liposomal PBS and L-Wnt3a=liposomalWnt3a. Arrowsmark the edge of intact bone. Scale bars: 40 mm (A) and (B); and 5 mm(F) and (G).

FIG. 6. Histological appearance of regenerated bone derived from L-Wnt3atreated aged bone grafts. Aniline blue staining of injury site (boxedarea) treated with aged bone marrow incubated in L-PBS (A) or L-Wnt3a(B). (C) Gomori trichrome staining of aged host's fatty bone marrowcavity, and the adjacent injury (D) area that received an L-PBS treatedaged bone graft; fibrous tissue is stained turquoise blue. (E) Gomoritrichrome staining of aged host's fatty bone-marrow cavity, and theadjacent injury (F) area that received an L-Wnt3a treated aged bonegraft; mature osteoid matrix stains dark turquoise and osteocyte nucleistain red. (G) Under polarized light, picrosirius red stainingidentifies fibrous tissue that has formed from aged bone graft treatedwith L-PBS. Compare with the osteoid matrix (H) that has formed fromaged bone graft treated with L-Wnt3a. Abbreviations: L-PBS=liposomalPBS, and L-Wnt3a=liposomal Wnt3a. Arrows mark the edge of intact bone.Scale bars: 500 μm (A) and (B); 100 μm (C) through (F); and 200 μm (G)and (H).

FIG. 7. Bone graft material contains stem and progenitor cellpopulations. (A) Gomori staining of BGM harvested from rat femur, (B)the iliac crest, and (C) the tibia. (D) Quantitative RTPCR analyses ofendogenous osteogenic gene expression in freshly harvested rat BGM fromthe indicated sources. (E) Schematic of experimental design, whereautologous BGM is transplanted into the SRC of rats. (F) Representativetissue sections of iliac crest BGM on post-transplant day 7, stained todetect BrdU incorporation. Dotted lines indicate trabecular bone chipsincluded in the BGM. (G) Runx2, (H) Sox9, and (I) PPAR□ expression. (J)Representative tissue sections of BGM stained with Aniline blue todetect osteoid matrix; asterisks indicate new bone matrix as opposed toold bone chips (yellow dotted line). The kidney surface is indicatedwith a dotted white line in this panel, and in G. (K) Safranin O/Fastgreen histology to detect proteoglycan-rich cartilage (red), and (L)Gomori trichrome staining to detect adipocytes. Abbreviations: BrdU,bromodeoxyuridine; PPAR□, peroxisome proliferator-activated proteingamma. Scale bars: 50 μm, asterisks: p<0.05.

FIG. 8. Bone graft material is Wnt responsive (A) GFP+ve cells inAxin2^(creERT2); R26m^(TmG) mice, visualized by immunostaining of theperiosteum and (B) endosteum. (C) Quantification of GFP^(+ve)cells/total cells within specified microscopic fields of view. (D)GFP^(+ve) cells in the BGM were visualized by fluorescence. (E)Quantitative absolute RT-PCR results for endogenous Axin2, Lef1, andGAPDH expression in BGM^(young) (green bars) and BGM^(aged) (grey bars).(F) Western blot analyses for Wnt3a, total beta catenin, Axin2, and betaactin in BGM^(young) (green bars) and BGM^(aged) (grey bars). Scalebars=50 μm. Asterisks: p<0.05.

FIG. 9. The osteogenic differentiation potential of BGM declines withage. (A) Quantitative RT-PCR analyses for expression of alkalinephosphatase, Osterix, and Osteocalcin in BGM^(young) (green bars) andBGM^(aged) (grey bars). (B) BGM harvested from ACTB-eGFP mice,transplanted into the SRC and visualized under brightfield and (C)fluorescent light to detect the GFP signal in BGM. (D) Representativetissue sections stained with Aniline blue (inset) from BGM^(young) (N=5)and (E) BGM^(aged) (N=5). Dotted line indicates the kidney surface. (F)Histomorphometric analyses of Aniline blue^(+ve) pixels within the totalarea occupied by the BGM on post-transplant day 7. (G) Representativetissue sections stained to detect ALP activity from BGM^(young) (N=5)and (H) BGM^(aged) (N=5). (I) Quantification of ALP^(+ve) pixels withinthe total area occupied by the BGM on post-transplant day 7. (J)Representative tissue sections immunostained for GFP from BGM^(young)(N=5) and (K) BGM^(aged) (N=5). (L) Quantification of GFP^(+ve) pixelswithin the total area occupied by the BGM on post-transplant day 7.Abbreviations: ALP, alkaline phosphatase; Oc, Osteocalcin. Scale bars:100 μm. Asterisks: p<0.05; double asterisks: p<0.01.

FIG. 10. Osteogenic differentiation of BGM requires an endogenous Wntsignal. (A) Representative tissue sections stained for ALP activity inBGM treated with the murine IgG2α Fc fragment (Ad-Fc) or (B) adenovirusexpressing the soluble Wnt antagonist Dkk1 (Ad-Dkk1). (C) Representativetissue sections immunostained for PPAR□ in BGM treated with Ad-Fc or (D)Ad-Dkk1. (E) Representative tissue sections immunostained for Dlk1 inBGM treated with Ad-Fc or (F) Ad-Dkk1. (G) Micro-CT reconstruction todetect bone formation in defect sites that received BGM treated withAd-Fc or (H) Ad-Dkk1. Original defect is indicated with a dotted redcircle. (I) New bone volume (N=5) calculated from micro-CT data±SEM. (J)Aniline blue staining on representative tissue sections from defectsites that received BGM treated with Ad-Fc or (K) Ad-Dkk1. (L)Quantification of new bone volume using histomorphometric analyses (seeMethods). (I) PPAR-□ expression in BM grafts treated with Ad-Fc or (J)Ad-Dkk1. Single asterisk p<0.05. Scale bars: A-B, 200 μm, CF, J-K, 50μm, G-H, 2 mm.

FIG. 11. Wnt3a activates BGM^(aged) and restores its osteogenicdifferentiation potential. (A) BGMs from aged ACTB-eGFP mice, treatedwith L-PBS or L-WNT3A (0.15 μg/ml) for 1 h then either assayed byqRT-PCR for target gene expression 24 h later, or immediatelytransplanted into the SRC for 7 days. (B) Fold change in Axin2 and Lef1expression in BGM^(aged) treated with either L-PBS (grey bars) orL-WNT3A (blue bars). (C) Western blot analysis of total beta catenin,Axin2, and beta actin in BGM^(aged) treated with either L-PBS (greybars) or L-WNT3A (blue bars). After harvesting BGM^(aged) from the SRCon post-transplant day 4, representative tissue sections from (D) L-PBS(N=5) and (E) L-WNT3A were stained for BrdU incorporation (N=5). (F)Quantification of BrdU^(+ve) pixels within a microscopic field of viewcentered in the middle of the bone grafts. (G) Representative tissuesections from L-PBS (N=5) and (H) L-WNT3A treated (N=5) samples, stainedfor BrdU incorporation on post-transplant day 7. (I) Quantification ofBrdU+ve pixels as above. (J) Representative tissue sections from L-PBS(N=5) and (K) L-WNT3A treated (N=5) samples, immunostained for Dlk1expression on post-transplant day 7. (L) Quantification of Dlk1+vepixels within the total area occupied by the BGM on post-transplant day7. (M) Representative tissue sections from L-PBS (N=5) and (N) L-WNT3Atreated (N=5) samples, immunostained for Oc expression onpost-transplant day 7. (O) Quantification of Oc^(+ve) pixels asdescribed for Dlk1. (P) Representative tissue sections stained withAniline blue to detect osteoid matrix in L-PBS (N=5) and (Q) LWNT3Atreated (N=5) samples. (R) Histomorphometric quantification of new bonematrix; see Methods for details. Abbreviations as in previous figurelegends. Scale bars: 100 μm. Asterisks: p<0.05; double asterisks:p<0.01.

FIG. 12. L-WNT3A stimulates BGM stem cells and improves spinal fusion(A) Human MSC cultures were treated with L-PBS or L-WNT3A at 37° C. forthe time points indicated and qRTPCR for Axin2 expression was used todetermine Wnt-response. (B) Murine SSC were treated with L-PBS or LWNT3Afor 12 h at 37° C. and Wnt response was assayed with qRT-PCR for Axin2expression. (C) Quantitative absolute RT-PCR analyses for Axin2 and Lef1expression in response to 1 h incubation at room temperature with L-PBS(dashed line) or L-WNT3A (0.15 μg/mL; blue line). Data is expressed as aratio of RNA copies/total RNA content over a 24 h period. (D) Ratspinous processes were exposed via minimal incisions and standardizedvolumes of autologous BGM from the iliac crest were treated with L-PBSor L-WNT3A for 1 hr then (E) transplanted between the transversesprocesses of the L4 and L5 vertebrae. (F) At POD2 Micro-CT acquisitionswere performed for graphs (pink) treated with L-PBS and (G) L-WNT3A. (H)At POD49 Micro-CT acquisitions were again performed to evaluate the bonegrowth of the transplants treated with L-PBS (gray) and (I) L-WNT3A(blue).(J) Transplant growth was graphed for each of the treatmentgroups as fold volume, comparing each graft size on POD2 to its size onPOD49 (indicated by the colors stated above). Abbreviations: L4, Lumbar#4, L5, Lumbar #5, AP, apical process, SP, spinous process, TP,transverse process, POD, postoperation day.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods described, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention will be limited only by the appended claims. Where a range ofvalues is provided, it is understood that each intervening value, to thetenth of the unit of the lower limit unless the context clearly dictatesotherwise, between the upper and lower limit of that range and any otherstated or intervening value in that stated range is encompassed withinthe invention. The upper and lower limits of these smaller ranges mayindependently be included in the smaller ranges encompassed within theinvention, subject to any specifically excluded limit in the statedrange. Unless defined otherwise, technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, N.Y. 1994), provides one skilled in the art with ageneral guide to many of the terms used in the present application.

All publications mentioned herein are expressly incorporated herein byreference to disclose and describe the methods and/or materials inconnection with which the publications are cited.

Wnt protein. Wnt proteins form a family of highly conserved secretedsignaling molecules that regulate cell-to-cell interactions duringembryogenesis. The terms “Wnts” or “Wnt gene product” or “Wnt protein”or “Wnt polypeptide” are used interchangeable and encompass nativesequence Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptidefragments and chimeric Wnt polypeptides. In some embodiments of theinvention, the Wnt protein comprises palmitate covalently bound to acysteine residue. A “native sequence” polypeptide is one that has thesame amino acid sequence as a Wnt polypeptide derived from nature,regardless of the method used for its production. Such native sequencepolypeptides can be isolated from cells producing endogenous Wnt proteinor can be produced by recombinant or synthetic means. Thus, a nativesequence polypeptide can have the amino acid sequence of, e.g. naturallyoccurring human polypeptide, murine polypeptide, or polypeptide from anyother mammalian species, or from non-mammalian species, e.g. Drosophila,C. elegans, and the like.

The term “native sequence Wnt polypeptide” includes, without limitation,human and murine Wnt polypeptides. Human Wnt proteins include thefollowing: Wnt1, Genbank reference NP005421.1; Wnt2, Genbank referenceNP003382.1, which is expressed in brain in the thalamus, in fetal andadult lung and in placenta; two isoforms of Wnt2B, Genbank referencesNP004176.2 and NP078613.1. Isoform 1 is expressed in adult heart, brain,placenta, lung, prostate, testis, ovary, small intestine and colon. Inthe adult brain, it is mainly found in the caudate nucleus, subthalamicnucleus and thalamus. Also detected in fetal brain, lung and kidney.Isoform 2 is expressed in fetal brain, fetal lung, fetal kidney, caudatenucleus, testis and cancer cell lines. Wnt 3 and Wnt3A play distinctroles in cell-cell signaling during morphogenesis of the developingneural tube, and have the Genbank references NP11 0380.1 and X56842(Swiss-Prot P56704), respectively.

The native human Wnt3A amino acid and nucleotide sequences arespecifically disclosed as SEQ ID NOs: 1 and 2, respectively. Wnt3A isexpressed in bone marrow. Wnt 4 has the Genbank reference NP11 0388.2.Wnt 5A and Wnt 5B have the Genbank references NP003383.1 and AK013218.Wnt 6 has the Genbank reference NP006513.1; Wnt 7A is expressed inplacenta, kidney, testis, uterus, fetal lung, and fetal and adult brain,Genbank reference NP004616.2. Wnt 7B is moderately expressed in fetalbrain, weakly expressed in fetal lung and kidney, and faintly expressedin adult brain, lung and prostate, Genbank reference NP478679.1. Wnt 8Ahas two alternative transcripts, Genbank references NP114139.1 andNP490645.1. Wnt 8B is expressed in the forebrain, and has the Genbankreference NP003384.1. Wnt 10A has the Genbank reference NP079492.2. Wnt10B is detected in most adult tissues, with highest levels in heart andskeletal muscle. It has the Genbank reference NP003385.2. Wnt 11 isexpressed in fetal lung, kidney, adult heart, liver, skeletal muscle,and pancreas, and has the Genbank reference NP004617.2. Wnt 14 has theGenbank reference NP003386.1. Wnt 15 is moderately expressed in fetalkidney and adult kidney, and is also found in brain. It has the Genbankreference NP003387.1. Wnt 16 has two isoforms, Wnt-16a and Wnt-16b,produced by alternative splicing. Isoform Wnt-16B is expressed inperipheral lymphoid organs such as spleen, appendix, and lymph nodes, inkidney but not in bone marrow. Isoform Wnt-16a is expressed atsignificant levels only in the pancreas. The Genbank references areNP057171.2 and NP476509.1. All GenBank, SwissProt and other databasesequences listed are expressly incorporated by reference herein.

The term “native sequence Wnt protein” or “native sequence Wntpolypeptide” includes the native proteins with or without the initiatingN-terminal methionine (Met), and with or without the native signalsequence. The terms specifically include the 352 amino acids long nativehuman Wnt3a polypeptide, without or without its N terminal methionine(Met), and with or without the native signal sequence.

A “variant” polypeptide means a biologically active polypeptide asdefined below having less than 100% sequence identity with a nativesequence polypeptide. Such variants include polypeptides wherein one ormore amino acid residues are added at the N- or C-terminus of, orwithin, the native sequence; from about one to forty amino acid residuesare deleted, and optionally substituted by one or more amino acidresidues; and derivatives of the above polypeptides, wherein an aminoacid residue has been covalently modified so that the resulting producthas a non-naturally occurring amino acid. Ordinarily, a biologicallyactive Wnt variant will have an amino acid sequence having at leastabout 90% amino acid sequence identity with a native sequence Wntpolypeptide, preferably at least about 95%, more preferably at leastabout 99%.

A “chimeric” Wnt polypeptide is a polypeptide comprising a Wntpolypeptide or portion (e.g., one or more domains) thereof fused orbonded to heterologous polypeptide. The chimeric Wnt polypeptide willgenerally share at least one biological property in common with a nativesequence Wnt polypeptide. Examples of chimeric polypeptides includeimmunoadhesins, combine a portion of the Wnt polypeptide with animmunoglobulin sequence, and epitope tagged polypeptides, which comprisea Wnt polypeptide or portion thereof fused to a “tag polypeptide”. Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made, yet is short enough such that it does notinterfere with biological activity of the Wnt polypeptide. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 6-60 amino acid residues.

A “functional derivative” of a native sequence Wnt polypeptide is acompound having a qualitative biological property in common with anative sequence Wnt polypeptide. “Functional derivatives” include, butare not limited to, fragments of a native sequence and derivatives of anative sequence Wnt polypeptide and its fragments, provided that theyhave a biological activity in common with a corresponding nativesequence Wnt polypeptide. The term “derivative” encompasses both aminoacid sequence variants of Wnt polypeptide and covalent modificationsthereof.

Biologically Active Wnt. The methods of the present invention providefor Wnt compositions that are active when administered to an animal,e.g. a mammal, such as a human, in vivo. One may determine the specificactivity of a Wnt protein in a composition by determining the level ofactivity in a functional assay, for example in an in vitro assay, orafter in vivo administration in a test model, e.g. accelerating boneregeneration, upregulation of stem cell proliferation, etc.,quantitating the amount of Wnt protein present in a non-functionalassay, e.g. immunostaining, ELISA, quantitation on Coomasie or silverstained gel, etc., and determining the ratio of in vivo biologicallyactive Wnt to total Wnt.

Lipid Structure. As used in the methods of the invention, lipidstructures are found to be important in maintaining the activity of Wntproteins following in vivo administration. The Wnt proteins are notencapsulated in the aqueous phase of these structures, but are ratherintegrated into the lipid membrane, and may be inserted in the outerlayer of a membrane. Such a structure is not predicted from conventionalmethods of formulating proteins in, for example, liposomes. A Wntpolypeptide with such lipid structure is referred herein as L-Wnt, suchas L-Wnt3a. The methods used for tethering Wnt proteins to the externalsurface of a liposome or micelle may utilize a sequence so as toemphasize the exoliposomal display of the protein, where crude liposomesare first pre-formed; Wnt protein is then added to the crude mixture,which will favor addition of exo-liposomal Wnt, followed by variousformulation steps, which may include size filtering; dialysis, and thelike. Suitable lipids include fatty acids, neutral fats such astriacylglycerols, fatty acid esters and soaps, long chain (fatty)alcohols and waxes, sphingoids and other long chain bases, glycolipids,sphingolipids, carotenes, polyprenols, sterols, and the like, as well asterpenes and isoprenoids. For example, molecules such as diacetylenephospholipids may find use. Included are cationic molecules, includinglipids, synthetic lipids and lipid analogs, having hydrophobic andhydrophilic moieties, a net positive charge, and which by itself canform spontaneously into bilayer vesicles or micelles in water. Liposomesmanufactured with a neutral charge, e.g. DMPC, are preferred. The termalso includes any amphipathic molecules that can be stably incorporatedinto lipid micelle or bilayers in combination with phospholipids, withits hydrophobic moiety in contact with the interior, hydrophobic regionof the micelle or bilayer membrane, and its polar head group moietyoriented toward the exterior, polar surface of the membrane.

The term “cationic amphipathic molecules” is intended to encompassmolecules that are positively charged at physiological pH, and moreparticularly, constitutively positively charged molecules, comprising,for example, a quaternary ammonium salt moiety. Cationic amphipathicmolecules typically consist of a hydrophilic polar head group andlipophilic aliphatic chains. Similarly, cholesterol derivatives having acationic polar head group may also be useful. See, for example, Farhoodet al. (1992) Biochim. Biophys. Acta 1111:239-246; Vigneron et al.(1996) Proc. Natl. Acad. Sci. (USA) 93:9682-9686. Cationic amphipathicmolecules of interest include, for example, imidazolinium derivatives(WO 95/14380), guanidine derivatives (WO 95/14381), phosphatidyl cholinederivatives (WO 95/35301), and piperazine derivatives (WO 95/14651).Examples of cationic lipids that may be used in the present inventioninclude DOTIM (also called BODAI) (Saladin et al., (1995) Biochem. 34:13537-13544), DDAB (Rose et al., (1991) BioTechniques 10(4):520-525),DOTMA (U.S. Pat. No. 5,550,289), DOTAP (Eibl and Wooley (1979) Biophys.Chem. 10:261-271), DMRIE (Feigner et al., (1994) J. Bioi. Chem. 269(4):2550-2561), EDMPC (commercially available from Avanti Polar Lipids,Alabaster, Ala.), DCC hoi (Gau and Huang (1991) Biochem. Biophys. Res.Comm. 179:280-285), DOGS (Behr et al., (1989) Proc. Nat!. Acad. Sci.USA, 86:6982-6986), MBOP (also called MeBOP) (WO 95/14651), and thosedescribed in WO 97/00241.

While not required for activity, in some embodiments a lipid structuremay include a targeting group, e.g. a targeting moiety covalently ornon-covalently bound to the hydrophilic head group. Head groups usefulto bind to targeting moieties include, for example, biotin, amines,cyano, carboxylic acids, isothiocyanates, thiols, disulfides,ahalocarbonyl compounds, a,p-unsaturated carbonyl compounds, alkylhydrazines, etc. Chemical groups that find use in linking a targetingmoiety to an amphipathic molecule also include carbamate; amide (amineplus carboxylic acid); ester (alcohol plus carboxylic acid), thioether(haloalkane plus sulfhydryl; maleimide plus sulfhydryl), Schiffs base(amine plus aldehyde), urea (amine plus isocyanate), thiourea (amineplus isothiocyanate), sulfonamide (amine plus sulfonyl chloride),disulfide; hyrodrazone, lipids, and the like, as known in the art. Forexample, targeting molecules may be formed by converting a commerciallyavailable lipid, such as DAGPE, a PEG-PDA amine, DOTAP, etc. into anisocyanate, followed by treatment with triethylene glycol diamine spacerto produce the amine terminated thiocarbamate lipid which by treatmentwith the para-isothiocyanophenyl glycoside of the targeting moietyproduces the desired targeting glycolipids. This synthesis provides awater soluble flexible linker molecule spaced between the amphipathicmolecule that is integrated into the nanoparticle, and the ligand thatbinds to cell surface receptors, allowing the ligand to be readilyaccessible to the protein receptors on the cell surfaces. Furtherinformation about liposomal Wnt compositions and their use is found inU.S. Application Publication 20120115788.

The term “bone graft” is used herein in the broadest sense andspecifically includes autografts and allografts, harvested from thepatient's own bones or from an individual other than the one receivingthe graft, including cadavers, respectively. The term “bone graft” alsoincludes autologous or allogeneic pluripotent stem cell populations,e.g. stem cells harvested from bone marrow, e.g. bone marrow-derivedmesenchymal stem cells. Bone grafts can be obtained from a donor byvarious means, including without limitation reamer, irrigation,aspirator methods.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Osteogenic competence is enhanced by incubating cells for a bone graftwith an effective dose of a Wnt protein, e.g. L-Wnt3A, for a period oftime sufficient to enhance osteogenic potential.

The bone graft material, as used herein, refers to a cellularcomposition obtained from a donor, which donor may be living orcadaveric. Bone graft material typically comprises complex cellpopulations, and includes stem cells such as mesenchymal stem cells, andmay also comprise osteocytes and progenitors thereof. The donor may beallogeneic or autologous relative to the recipient. The quantity ofcells for a bone graft may vary with the donor, the recipient, purposeof graft, and the like. A bone graft may comprise up to about 10³, up toabout 10⁴, up to about 10⁵, up to about 10⁶, up to about 10⁷, up toabout 10⁸, up to about 10⁹, up to about 10¹⁰ or more cells.

The bone graft material is obtained from the donor, for example from theiliac crest, from the mandibular symphysis (chin area), from reaming,aspirating, and irrigating the femur and/or tibia, fibula, ribs,anterior mandibular ramus; parts of spinal bone, e.g. those removedduring surgery, cadaver bones, etc. The graft material may be bonemarrow, for example scraped from the endosteal surface of a suitablebone, or may be a block graft containing marrow and a small block ofbone. Allograft bone can be taken from cadavers, bone banks, etc. forexample sing a femoral head from hip replacement surgery. The bone graftmaterial can be used fresh, or can be cryo-preserved as known in the artuntil it is needed.

The cells of the bone graft are suspended in a suitable culture mediumin the presence of an effective dose of a liposomal Wnt protein, e.g.L-Wnt3A. Any suitable medium can be used, e.g. DMEM, RPMI, PBS, etc.Cells are typically resuspended at a concentration that maintainsviability during the incubation procedure, e.g. up to about 10⁴/ml, upto about 10⁵/ml, up to about 10⁶/ml, up to about 10⁷/ml. The incubationtemperature is usually not more than about 37° C., and may be lower,e.g. up to about 32° C., up to about 25° C., up to about 15° C., up to10° C., up to about 4° C., but typically above freezing unlessspecifically prepared for cryopreservation.

The effective dose of the Wnt protein may vary depending on the source,purity, preparation method, etc. Where the Wnt protein is L-Wnt3A, theeffective dose is usually at least about 0.1 μg/ml, at least about 0.5μg/ml, at least about 1 μg/ml, at least about 2.5 μg/ml, at least about5 μg/ml, at least about 7.5 μg/ml, at least about 10 μg/ml, at leastabout 15 μg/ml, and may be at least about 25 vg/ml, at least about 50μg/ml, at least about 100 μg/ml.

The bone graft material is incubated with the Wnt protein for a periodof time sufficient to enhance osteogenic capacity. The enhancement canbe measured in various ways, e.g. by increased expression of Axin2, byincreased mitotic activity in the bone graft material (measured at fromabout day 2 to about day 6 post-transplantation; by increased boneformation post-transplantation, by increased expression of Runx2 orOsteocalcin, by reduced apoptosis post-transplantation; or by volume ofbone produced post-transplantation. The volume of increased bone may beabout 1.5-fold, about 2-fold, about 3-fold or more relative to thevolume that would be obtained in the absence of wnt treatment.

The bone graft material is usually contacted with the Wnt protein for atleast about 1 hour, at least about 2 hours, and up to about 36 hours, upto about 24 hours, up to about 18 hours, up to about 15 hours, up toabout 12 hours, up to about 8 hours, up to about 6 hours, up to about 4hours.

Following incubation, the bone graft material may be transplanted into arecipient following conventional protocols, e.g. for repair of spinalbone, fractures, dental supports, and the like.

Osteogenic capacity is particularly restored to aged bone grafts byincubation with the a Wnt protein. Initially, liposomal Wnt3a treatmentreduces cell death in aged bone grafts. Later after transplantation,bone grafts treated with liposomal Wnt3a gave rise to significantly morebone (p<0.05). As it will be apparent from the examples, liposomal Wnt3atreatment enhanced cell survival in the graft and re-established thebone-forming ability of grafts from aged animals.

Accordingly, the present invention provides a safe, effective, andclinically applicable regenerative medicine-based strategy forrevitalizing bone grafts from aged patients, and from other patientswith diminished healing potential, such as, for example, smokers,diabetics, or patients, with nutritional deficits.

All scientific and patent publications, patents and patent applicationscited in this specification are herein incorporated by reference as ifeach individual publication were specifically and individually indicatedto be incorporated by reference. Further details of the invention areprovided in the following non-limiting Examples.

Example I Wnt3a Reestablishes Osteogenic Capacity to Bone Grafts fromAged Animals

Age-related fatty degeneration of the bone marrow contributes to delayedfracture-healing and osteoporosis related fractures in the elderly. Themechanisms underlying this fatty change may relate to the level of Wntsignaling within the aged marrow cavity. In youth, long bones are filledwith heme-rich marrow; with age, this is replaced by fatty marrow.Age-related fatty degeneration of the marrow is strongly associated withdelayed skeletal healing and osteoporosis-related fractures in theelderly, which constitutes a growing biomedical burden. Consequently,considerable effort has gone into understanding the conversion of bonemarrow into a predominantly fatty tissue. This fatty degeneration of thebone marrow occurs in parallel with a loss in osteogenic potential,which is revealed when marrow is used clinically for bone graftingpurposes.

A patient's own bone and marrow is considered the “gold standard”, butthese autografts are oftentimes inadequate when the patient is elderly.There are at least multiple, distinct stem/progenitor cell populationsthat reside in the bone marrow cavity, including mesenchymal stem cells(MSCs). Although MSCs can give rise to cartilage, bone, fat, and musclecells when cultured in vitro, MSCs residing in the marrow cavity itselfonly differentiate into an osteogenic or an adipogenic lineage, andgrowing evidence indicates that this adipogenic-osteogenic fate decisionis regulated by beta catenin-dependent Wnt signaling. For example,enhancing Wnt signaling, by activating mutations in the Wnt LRP5receptor, causes a high bone mass phenotype in humans. In vitro, thissame activating mutation represses adipocyte differentiation of humanmesenchymal stem cells. On the other hand, reduced Wnt signaling, forexample in the osteolytic disease multiple myeloma, is associated withaggressive bone loss and a concomitant increase in marrow adiopogenesisat the expense of hematopoiesis. Together these observations support ahypothesis that Wnt signaling has a positive role in stimulatingosteogenesis and inhibiting adipogenesis.

Transgenic mice were used in conjunction with a syngeneic bone graftmodel to follow the fates of cells involved in the engraftment.Immunohistochemistry along with quantitative assays were used toevaluate Wnt signaling and adipogenic and osteogenic gene expression inbone grafts from young and aged mice. Liposomal Wnt3a protein (L-Wnt3a)was tested for its ability to restore osteogenic potential to aged bonegrafts in critical size defect models created in mice and in rabbits.Radiography, micro-CT reconstruction, histology, and histomorphometricmeasurements were used to quantify bone healing resulting from L-Wnt3aor control, L-PBS treatment. Gene expression profiling of bone graftsdemonstrated that aging was associated with a shift away from anosteogenic profile and towards an adipogenic one. This age-relatedadipogenic shift was accompanied by significantly reduced Wnt expressionand Wnt activity (p<0.05) in bone grafts from aged animals.

Transgenic mice were used in conjunction with a syngeneic bone-graftmodel to follow the fates of cells involved in the engraftment.Immunohistochemistry along with quantitative assays were used toevaluate Wnt signaling and adipogenic and osteogenic gene expression inbone grafts from young and aged mice. Liposomal Wnt3a protein (L-Wnt3a)was tested for its ability to restore osteogenic potential to aged bonegrafts in critical-size defect models created in mice and in rabbits.Radiography, microquantitative computed tomography (micro-CT)reconstruction, histology, and histomorphometric measurements were usedto quantify bone-healing resulting from L-Wnt3a or a control substance(liposomal phosphate-buffered saline solution [L-PBS]).

Expression profiling of cells in a bone graft demonstrated a shift awayfrom an osteogenic gene profile and toward an adipogenic one with age.This age-related adipogenic shift was accompanied by a significantreduction (p<0.05) in Wnt signaling and a loss in osteogenic potential.In both large and small animal models, osteogenic competence wasrestored to aged bone grafts by a brief incubation with the stem-cellfactor Wnt3a. In addition, liposomal Wnt3a significantly reduced celldeath in the bone graft, resulting in significantly more osseousregenerate in comparison with controls.

Liposomal Wnt3a enhances cell survival and reestablishes the osteogeniccapacity of bone grafts from aged animals in an effective, clinicallyapplicable, regenerative medicine-based strategy for revitalizing bonegrafts from aged patients.

Materials and Methods

Animals.

All procedures were approved by the Stanford Committee on AnimalResearch. Axin2^(LacZ/+) mice have been described. Beta-actin-enhancedgreen fluorescent protein (ACTB-eGFP) transgenic mice (The JacksonLaboratory, Sacramento, Calif.) were chosen because of robust expressionlevels of GFP in bone, marrow, and other relevant cell populations.ACTB-eGFP transgenic mice were crossed with Axin2^(LacZ/+) mice toobtain Axin2^(LacZ/+), Axin2^(LacZ/+)/ACTB-eGFP, ACTB-eGFP and wild-type(WT) mice; twelve to sixteen weeks old mice were considered young; micegreater than forty weeks of age were considered aged. Aged (eightmonths) New Zealand white rabbits were used. One rabbit served as thebone graft donor, and nine rabbits served as experimental animals.

Bone-Grafting in Mice.

Host mice (male only) were anesthetized by intraperitoneal injection ofketamine (80 mg/kg) and xylazine (16 mg/kg). A 3-mm incision was made toexpose the parietal bone; a circumferential, full-thickness defect witha 2-mm diameter was created with use of a micro dissecting trephine; thedura mater was not disturbed. Bone graft was harvested from the femoraand tibiae, pooled, and divided into aliquots. Each 20-μL aliquot wasincubated in 10 μL of Dulbecco modified Eagle Medium (DMEM) with 10%fetal bovine serum (FBS) containing liposomal phosphate-buffered salinesolution (L-PBS) or liposomal Wnt3a protein (L-Wnt3a) (effectiveconcentration=0.15 μg/mL) at 37° C. while the calvarial defect wasprepared. Bone grafts were transplanted to the calvarial defect, and theskin was closed.

Bone-Grafting in Rabbits.

Host rabbits were anesthetized with a subcutaneous injection ofglycopyrrolate (0.02 mg/kg) and buprenorphine (0.05 mg/kg), anintramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg),and an intravenous injection of cefazolin (20 mg/kg), and maintainedunder 1% to 3% isoflurane. A 2.5-cm incision was made, the ulnar borderwas visualized, and a 1.5-cm segmental defect was created with anoscillating saw (Stryker System 5, Kalamazoo, Mich.). The segment wasremoved along with its periosteal tissues. Bone graft was harvested fromthe pelvis and femur, pooled, and divided into aliquots. Eachapproximately 400-mg aliquot was combined with L-PBS (500 μL) or L-Wnt3a(effective concentration=0.5 μg/mL) and kept on ice on the back tablewhile the ulnar defect was created in host rabbits. Bone grafts weretransplanted to the ulnar defect, and the muscle and skin were closed.The procedure was performed bilaterally (i.e., both sides eitherreceived L-PBS or L-Wnt3a). This approach eliminated the possibility,however remote, that the bone graft would have an unanticipated systemiceffect.

In Vitro Wnt Stimulation of Rabbit Bone Marrow.

Bone marrow from aged rabbits was incubated with L-PBS or L-Wnt3a(effective concentration=0.15 μg/mL) at 37° C. for twelve hours. TotalDNA was assayed with use of PicoGreen dsDNA kit (Life Technologies,Carlsbad, Calif.) to ensure that grafts had equivalent cell volumes.Caspase activity was assayed with use of a standard kit (RocheDiagnostics, Indianapolis, Ind.).

Tissue Preparation.

Immediately after euthanasia (time points specified in each experiment),the entire skeletal element, including muscle, connective tissue, and/ordura was harvested, removed of its epidermis, and fixed in 4%paraformaldehyde at 4° C. for twelve hours. Samples were decalcified in19% EDTA (ethylenediaminetetraacetic acid) before embedding in paraffin,or in optimal cutting temperature (OCT) compound. Sections were 10-μmthick.

Histology, Immunohistochemistry, and Histomorphometric Analyses.

Immunohistochemistry was performed as previously described. Antibodiesused included rabbit polyclonal anti-green fluorescent protein(anti-GFP) (Cell Signaling Technology, Danvers, Mass.), rabbitpolyclonal anti-DLK1 (EMD Millipore, Billerica, Mass.), anti-peroxisomeproliferator activated receptor-μ (anti-PPAR-μ) (Millipore), andanti-Ki67 (ThermoFisher Scientific, Waltham, Mass.). Thebromodeoxyuridine (BrdU) (Invitrogen, Camarillo, Calif.) and terminaldeoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (RocheDiagnostics) assays were performed following the manufacturers'instructions.

Movat pentachrome, aniline blue, Xgal, and alkaline phosphatase (ALP)stainings were performed as previously described. Tissue sections werephotographed with use of a Leica DM5000B digital imaging system (LeicaMicrosystems, Wetzlar, Germany). A minimum of five tissue sections persample was used for histomorphometric analyses.

Micro Quantitative Computed Tomography (Micro-CT) Analyses.

Mice were anesthetized with 2% isoflurane and scanned with use of amultimodal positron emission tomography-computed tomographydata-acquisition system (Inveon PET-CT; Siemens, Erlangen, Germany) at40-mm resolution. Data were analyzed with MicroView software (GEHealthcare, Chicago, Ill.). The three-dimensional region-of-interesttool was used to assign the structure and bone volume for each sample.

Assessment of the regenerate bone volume fraction (the percentagecalculated by dividing the total bone volume by the regenerate bonevolume [BV/TV, %]) was performed with use of high-resolution micro-CT(vivaCT 40; Scanco Medical, Br⁻uttisellen, Switzerland) and with 70 kVp,55 μA, 200-ms integration time, and a 10.5-μm isotropic voxel size. Theregion of interest was 2 cm in length and began 250 μm proximal to theedge of the defect and extended 250 μm distally beyond the opposing edgeof the defect (1.5 cm total diameter). Bone was segmented from softtissue with use of a threshold of 275 mg/cm³ hydroxyapatite. Scanningand analyses adhered to published guidelines.

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR).

Tissue samples were homogenized in TRIzol solution (Life Technologies).RNA was isolated (RNeasy; Qiagen, Germantown, Md.) and reversetranscription was performed (SuperScript III Platinum Two-Step qRT-PCRKit, Life Technologies) as described previously.

Statistical Analyses.

Results are presented as the mean plus the standard deviation, with “n”signifying the number of samples analyzed. Significant differencesbetween data sets were determined with use of two-tailed Student t testsand nonparametric Wilcoxon tests. Significance was attained at p<0.05,and all statistical analyses were performed with GraphPad Prism software(GraphPad Software, San Diego, Calif.).

Results

Bone-Marrow Grafts have Osteogenic Potential.

To follow the fate of the bone-graft material, we harvested whole bonemarrow from ACTB-eGFP transgenic mice, subdivided it intoequivalent-size aliquots (FIG. 1-A), then transplanted it into anonhealing, critical-size skeletal defect that was created in thecalvarium of syngeneic host mice (FIG. 1-B). The viable grafted cellsand their progeny were identifiable within the injury site by their GFPlabel (FIG. 1-C). When the donor and host were not geneticallyidentical, most of the grafted cells died; for that reason, onlysyngeneic, immunologically compatible donor-host combinations were used.

On post-graft day 1, GFP-positive cells, along with stromal tissue fromthe GFP-positive donor, occupied the injury site (FIG. 1-C). On day 5,BrdU staining confirmed the robust proliferation of cells in the defectsite (FIG. 1-D). On day 7, GFP immunostaining confirmed that graftedcells, or their progeny, remained at the defect site (FIGS. 1-E and1-F). The grafted cells and/or their progeny eventually differentiateinto osteoblasts and heal the defect (FIGS. 1-H and 1-J); in the absenceof a bone graft, the defect will not heal (FIGS. 1-G and 1-I).

Aged Bone Grafts Exhibit Fatty Degeneration.

With aging, human bone marrow undergoes fatty degeneration and a loss inosteogenic potential. A comparable age-related change is observed inmice, in which the gross appearance of murine bone marrow changes from aheme-rich, fat-free tissue in young animals to a fatty marrow in agedanimals. Quantitative RT-PCR analyses of the heterogeneous cellpopulation that constitutes a bone graft showed that relative to samplesfrom young animals, samples from aged animals showed significantlyhigher expression of the adipogenic genes fatty acid-binding protein 4(Fabp4) (p<0.01) and peroxisome proliferator-activated receptor gamma(PPAR-□) (p<0.01). Simultaneous with this adipogenic shift, bone graftsfrom aged mice also showed significantly reduced expression levels ofthe osteogenic genes ALP (p<0.05), osteocalcin (p<0.01), and osterix(p<0.05). Thus, fatty degeneration of the bone marrow observed in humansis recapitulated in mice at both a gross morphologic level and at aquantifiable, molecular level.

Fatty Degeneration is Associated with Reduced Osteogenic Potential in aBone Graft.

Compared with the osteogenic capacity of grafts from young animals,grafts from aged animals generated significantly less new bone (FIGS.2-A and 2-B; quantified in 2-C; p<0.05). This age-related reduction inosteogenic potential was not directly attributable to differences inengraftment efficiency. Using GFP immunostaining to identify the graftedcells, the distribution and number of GFP-positive cells was nearlyequivalent between bone grafts from young and aged mice (FIGS. 2-D and2-E; quantified in 2-F). Nor was the age-related alteration inosteogenic potential attributable to differences in the expansion of thegraft: Using both BrdUincorporation (FIGS. 2-G and 2-H) and qRT-PCR forproliferating cell nuclear antigen (PCNA) (FIG. 2-I) we found nearlyequivalent levels of cell proliferation in bone grafts from young andaged animals.

We gained insights into the basis for fatty degeneration and loss inosteogenic potential of aged bone grafts when we assessed the expressionlevel of nineteen mammalian Wnt genes in marrow cells. A subset of Wntgenes were weakly expressed in bone marrow from aged animals comparedwith young animals (p<0.05; FIG. 3-A). This reduction in Wnt geneexpression was paralleled by a reduction in Wnt responsiveness, asmeasured by significantly decreased expression of the Wnt direct targetgenes Tcf4, Lef1, and Axin2 (p<0.05; FIG. 3-B). These resultsdemonstrate that Wnt signaling is reduced in aged bone marrow.

L-Wnt3a Restores Osteogenic Capacity to Bone Grafts from Aged Mice.

The first Wnt protein to be purified was Wnt3a. Wnt3a acts via the“canonical” or beta-catenin dependent pathway and is a well-knownosteogenic stimulus. Given the reduced Wnt signaling in aged bonemarrow, we wondered if the addition of exogenous Wnt protein would besufficient to reestablish the osteogenic potential of bone graftsderived from aged animals.

All vertebrate Wnt proteins are hydrophobic; without a carrier, thehydrophobic Wnt3a rapidly denatures and becomes inactive. We solved thisin vivo delivery problem by packaging the hydrophobic Wnt3a in lipidparticles. This formulation of the human Wnt3a protein, liposomal Wnt3a(L-Wnt3a), is stable in vivo and promotes robust bone regeneration in amodified fracture model. Although exogenously applied Wnt3a has greatpotential as a therapeutic protein, safety remains a primary concern.The delivery of high concentrations of potent growth factors to askeletal injury site carries with it potential oncological risk to thepatient. To circumvent issues associated with prolonged or uncontrolledexposure to a growth factor, we delivered L-Wnt3a ex vivo. This wasaccomplished by incubating the aged bone graft with L-Wnt3a (n=30)immediately after harvest, while the recipient site was prepared.Control bone grafts were exposed to L-PBS (n=30) for the same duration.

Compared with aged grafts treated with L-PBS (FIG. 4-A), aged graftstreated with L-Wnt3a showed a dramatic enhancement in new bone formation(FIG. 4-B). Within seven days, defect sites that receivedL-Wnt3a-treated grafts had twofold more new bone than sites thatreceived L-PBS treated grafts (FIG. 4-C). By day 12, L-Wnt3a-treatedgrafts had 1.5-fold more new bone compared with L-PBS treated grafts(FIGS. 4-D and 4-E; quantified in 4-C).

Bone-Marrow-Derived Stem Cells are Wnt Responsive.

To gain insights into which cell population(s) in the bone graftresponded to the Wnt stimulus, we assayed different fractions of themarrow for Wnt responsiveness. In whole bone marrow, Wnt responsivenesswas below detectable levels. We separated whole bone marrow into anon-adherent population; once again Wnt responsiveness was below thelimit of detection (FIG. 4-F). In the adherent population, however,which contains connective tissue progenitor cells 53, 54, Wntresponsiveness was detected (FIG. 4-F). We then used establishedprotocols to further enrich for bone-marrow stem and/or stromal cellsfrom the attached population. Using immunostaining for CD45(−), CD73(+),CD105(+), and Stro1(+), we confirmed that this population was enrichedfor marrow-derived stem cells (FIG. 4-G). Relative to PBS-treatedCD45(−), CD73(+), CD105(+), and Stro1(+) cells, the Wnt3a-treatedpopulation showed a tenfold increase in Wnt responsiveness (FIG. 4-H).

We also monitored Wnt responsiveness in bone grafts using Xgal stainingof marrow from Axin2^(LacZ/+) mice. Very few Xgal^(+ve) cells were foundin aged bone grafts (FIG. 4-I) but Xgal^(+ve) cells were plentiful inyoung bone grafts (FIG. 4-K). Aged bone grafts were capable ofresponding to an L-Wnt3a stimulus, as shown by the increase inXgal^(+ve) cells following exposure (FIG. 4-J). Because the prevalenceof stem cells in the murine marrow cavity is quite low, it is likelythat the Wnt responsive population included more cells than the CD45(−),CD73(+), CD105(+), and Stro1(+) population.

L-Wnt3a Prevents Apoptosis in Bone Grafts.

The robust bone-inducing capacity of L-Wnt3a prompted us to extend ourstudies into a large animal, long-bone model. As in humans, aged rabbitsexperience fatty degeneration of their marrow. We utilized acritical-size ulnar defect model and transplanted aged bone grafts thathad been incubated with L-PBS or L-Wnt3a into the defect. We first notedthat when bone graft is harvested there is extensive programmed celldeath throughout the aggregate (FIG. 5-A). The addition of L-Wnt3asignificantly reduced this graft apoptosis (p<0.05) (FIG. 5-B). Weverified this pro-survival effect of L-Wnt3a, using caspase activity asa measure of cell apoptosis. L-Wnt3a significantly reduced caspaseactivity in cells of the bone graft (p<0.05; FIG. 5-C).

L-Wnt3a Potentiates the Osteogenic Capacity of Aged Bone Grafts. L-Wnt3aand L-PBS-treated rabbit bone grafts were introduced into the criticalsize defect and regeneration was assessed at multiple time points.Radiographic assessment at four weeks revealed the presence of abridging callus in sites that had received L-Wnt3a-treated graft (FIG.5-E); in comparison, sites that received L-PBS-treated bone graft showedminimal callus formation (FIGS. 5-D).

At eight weeks, micro-CT analyses demonstrated a persistent gap in sitesthat were treated with L-PBS bone grafts (FIG. 5-F) whereas sitestreated with L-Wnt3a bone graft exhibited robust bone formation (FIG.5-G). Histomorphometric analyses confirmed a significant differencebetween the two groups, both in bone volume and in bone volume dividedby total volume (FIG. 5-H).

We assessed the quality of the bone regenerate. Compared with controls(FIG. 6-A), L-Wnt3a-treated injury sites were filled with new bone (FIG.6-B). The bone marrow of the host rabbits had undergone fattydegeneration (FIG. 6-C), and a similar appearance was noted in theL-PBS-treated regenerate (FIG. 6-D). In the L-Wnt3a treated samples(FIG. 6-E), the host bone marrow showed a similar level of fattydegeneration as seen in the control animals, but the regenerate fromL-Wnt3a bone graft was woven bone (FIG. 6-F) and was distinguishablefrom the preexisting lamellar bone by both its location in the segmentaldefect site and its woven appearance. Under polarized light, picrosiriusred staining distinguished the mature, osteoid tissue found in theL-Wnt3a-treated bone grafts (FIG. 6-H) from the fibrous tissue of theL-PBS treated bone grafts (FIG. 6-G).

Stem-Cell and/or Progenitor Cell Populations in Bone Grafts.

The mammalian bone-marrow cavity is a functional niche that supportsmultiple stem-cell and/or progenitor cell populations. Marrow-derivedbone grafts, which are heterogeneous by nature, contain multiplepopulations, including some stem cells and/or progenitor cells. Thecontribution of these stem cells and/or progenitor cells to an osseousregenerate, however, remains unknown. Multiple marrow-derived stem-cellpopulations are Wnt-responsive and, using established protocols, weconfirmed that at least the CD45(−), CD73(+), CD105(+), and Stro1(+)stem-cell and/or stromal-cell population in the bone marrow isWnt-responsive (FIG. 4).

Wnt Signaling and Age-Related Fatty Degeneration of the Marrow.

In vitro, the abrogation of Wnt signaling causes mesenchymal stem cellsto differentiate into adipocytes whereas potentiation of Wnt signalingcauses mesenchymal stem cells to differentiate into osteoblasts. Thishas direct clinical relevance: With age, human bone marrow undergoesfatty degeneration and loses its osteogenic potential. Our data showthat this loss in osteogenic potential of aged bone grafts rests, inpart, on a reduced level of Wnt signaling: Compared with bone graftsfrom young mice, aged bone grafts show a dramatic reduction in Wnt geneexpression and Wnt responsiveness (FIG. 3). Adding L-Wnt3a to aged bonemarrow reestablishes its bone forming capacity (FIGS. 4, 5, and 6).

Conditions associated with decreased mobility, such as extended bed restand osteoporosis, are also associated with fatty degeneration of themarrow. Some data suggest that fatty degeneration is reversible, atleast experimentally. Clearly, understanding the basis for thisdegeneration—and the extent to which age-related changes in the skeletoncan be reduced—will be of considerable importance in devising newtreatment for bone injuries in elderly patients.

Growth-Factor-Augmented Bone Regeneration: Safety First.

Safety concerns have recently arisen surrounding the use of growthfactors to augment skeletal healing. Growth factor stimuli are largelythought to induce the proliferation of cells residing in the injurysite; because uncontrolled proliferation is a characteristic ofoncogenic transformation, this proliferative burst must be controlledboth spatially and temporally. For this reason, we designed an approachthat would limit whole-body exposure to L-Wnt3a. The targeted cells arethose in the bone graft itself, which is incubated with L-Wnt3a ex vivo.The activated cells—rather than the growth factor itself—are thenreintroduced into a defect site. This ex vivo approach restricts theL-Wnt3a stimulus spatially (to the graft itself, and not to hosttissues) and temporally (exposure to the Wnt stimulus only occurs duringthe incubation period). This ex vivo approach is tailored to clinicaluse and does not require a second procedure. Thus, packaging Wnt proteininto lipoparticles constitutes a viable strategy for the treatment ofskeletal injuries, especially those in individuals with diminishedhealing potential.

Example 2 Reengineering Autologous Bone Grafts with the Stem CellActivator WNT3A

Autologous bone grafting is the most commonly used procedure to treatbone defects, but is considered unreliable in elderly patients. Theefficacy of an autograft can be traced back to multipotent stem cellsresiding within the material. Aging attenuates the viability andfunction of these stem cells, leading to inconsistent rates of bonyunion. We show that age related changes in autograft efficacy areaccompanied by a loss of endogenous Wnt signaling in the material. Wemimicked this loss of endogenous Wnt signaling by overexpressing the Wntinhibitor Dkk1 and found that Wnt signaling is necessary for theosteogenic differentiation of an autograft. We developed an ex vivo drugdelivery system in which autografts were incubated in a stabilizedformulation of WNT3A protein then introduced it in vivo. Thebioengineered autograft showed significantly improved survival in thehosting site. Mitotic activity and osteogenic differentiation weresignificantly enhanced in MINT-activated autografts compared toautografts alone. In a spinal fusion model, aged autografts treated withL-WNT3A showed superior bone forming capacity compared to autograftsalone. Thus, a brief incubation in L-WNT3A reliably improves autologousbone grafting efficacy, which has the potential to significantly improvepatient care in the elderly.

The most common treatment for non-unions, delayed unions and posteriorcervical spine fusions is autologous bone grafting, or autografting.Autografts are successful in the vast majority of cases but the basisfor their bone forming (osteogenic) capacity is not entirely clear.Autografts are a heterogeneous collection of marrow blood products,connective tissue stroma, bony extracellular matrix, and a variety ofhematopoietic, vascular, and osteogenic stem cell populations and theyhave variously been described as osteoinductive, osteoconductive, andosteogenic. These terms, however, only describe cellular processes; theydo not provide insights into the basis for the osteogenic potential ofautografts.

Autografts become unreliable in older patients, and there are likely tobe multiple contributing factors for this degenerative state. Some datasuggests that the osteogenic capacity of autografts is dependent uponthe presence of stem or progenitor cells contained within the bone graftmaterial, and stem cell numbers are thought to decline with age in partbecause of accumulated DNA damage that ultimately results in cell cyclearrest and apoptosis. Other data argue that rather than a decline in thenumber of stem cells, their function deteriorates with age. Aged stemcells may also be less responsive to growth factor stimuli in theirenvironments; likewise, local or systemic levels of these growth factorstimuli may decline in the elderly. Aging also impacts the mitoticcapacity of stem cells: Stem cell senescence increases with age, in partbecause of a reduction in telomerase activity and subsequent telomereshortening. These reductions in stem cell function constrain thenormally robust regenerative responses of tissues such as the intestine,and muscle. Aging also impacts the mitotic capacity of stem cells.Similar mechanisms may be responsible for the loss in osteogeniccapacity of autografts.

Here, we tested the hypotheses that the osteogenic potential of anautograft is attributable to osteogenic stem cells in the graftmaterial; that aging impacts the Wnt responsive status of thesestem/progenitor populations; and that MINT-mediated activation of thestem cells can restore bone forming potential to autografts from elderlyanimals.

Methods

Animal care. All procedures followed protocols approved by the StanfordCommittee on Animal Research. Beta-actin-enhanced green fluorescentprotein (ACTB-eGFP; The Jackson Laboratory, Sacramento, Calif.) and CD1wild type, syngeneic mice were used. Mice<3 months old were consideredyoung; mice>10 months were considered aged. Axin2^(CreERT/+); R26RmTmG/+mice were purchased from Jackson Labs. Aged wild type Lewis rats(“retired breeders” from Charles Rivers, Mass.), were utilized forspinal fusion surgeries according to AAUC and IUPAC guidelines (protocol13146).

Collection and treatment of bone graft material (BGM) for rodent models.Both rats and mice were employed in this study. The use of mouse modelsallows for a broad spectrum of molecular analyses, however, becauseautografts are highly invasive for these small animals, we used ratswhen performing autografts (e.g., FIGS. 7 and 12), and syngeneic micewhen employing advanced molecular techniques (FIGS. 8-11). In all cases,bone graft material (BGM) was harvested from femurs, tibiae and iliaccrest by splitting the bones lengthwise, gently scraping the endostealsurface with a sharp instrument, and irrigating the marrow contents intoa collection dish. This method mimicked the RIA technique used inhumans.

To induce recombination in Axin2^(CreERT/+); R26R^(mTmG/+) mice (FIG.8), animals were given 160 μg/g body weight tamoxifen via IP injectionor gavage for 5 consecutive days. Tissues were harvested 7 days afterthe first treatment and analyzed by GFP immunostaining or fluorescence.

To ensure that BGM aliquots for transplantation into the SRC wereequivalent in terms of cellular content, BGM from 3 mice (littermates)was pooled then divided into 20 μL aliquots just as in the transplantassays. DNA content was extracted with the DNeasy Tissue Kit (QIAGEN)and relative DNA concentration was measured using the Quant-iT PicoGreendsDNA Kit (Invitrogen) and microplate fluorescence reader (BERTHOLD, BadWildbad, Germany). The percent variation in DNA content was <20%. Toobtain BGM^(ACT), freshly harvested BGM was placed into 20 μL of culturemedium containing a liposomal formulation of either phosphate-bufferedsaline (L-PBS) or WNT3A (L-WNT3A, effective L-WNT3A concentration=0.15μg/mL) and maintained at 23° C. for 1 hour.

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR).

Tissue samples were homogenized in TRIzol solution (Invitrogen). RNA wasisolated (RNeasy; Mini Kit; QIAGEN, MD, USA) and reverse transcriptionwas performed (SuperScript III First-Strand Synthesis Supermix forqRT-PCR, Invitrogen) as described previously. Quantitative real-time PCRwas carried out using Prism 7900HT Sequence Detection System (AppliedBiosystems, Foster City, Calif., USA) and Power SYBR Green PCR MasterMix (Applied Biosystems). Levels of gene expression were determined bythe CT method and normalizing to their GAPDH values. All reactions wereperformed in triplicate, means and standard deviations were calculated.Primers sequences (5′ to 3′) are as follows: Axin2,[for-TCATTTTCCGAGAACCCACCGC (SEQ ID NO:3)],[rev-GCTCCAGTTTCAGTTTCTCCAGCC (SEQ ID NO:4)]; Lef1,[for-AGGAGCCCAAAAGACCTCAT (SEQ ID NO: 5)], [rev-CGTGCACTCAGCTATGACAT(SEQ ID NO:6)]; GAPDH, [for-ACCCAGAAGACTGTGGATGG (SEQ ID NO:7)][rev-GGATGCAGGGATGATGTTCT (SEQ ID NO:8)]; ALP [for-ACCTTGACTGTGGTTACTGC(SEQ ID NO:9)], [rev-CATATAGGATGGCCGTGAAGG (SEQ ID NO:10)]; Osterix,[for-GGAGACCTTGCTCGTAGATTTC (SEQ ID NO:11)], [rev-GGGATCTTAGTGACTGCCTAAC(SEQ ID NO:12)]; Osteocalcin, [for-TGTGACGAGCTATCAAACCAG (SEQ ID NO:13)], [rev-GAGGATCAAGTTCTGGAGAGC (SEQ ID NO:14)].

Western Analyses.

BGM was harvested from young (N=5) and aged (N=5) mice and then placedin Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal BovineSerum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, andincubated at 37° C. in 5% CO2. After 24 hours, non-adherent cells wereremoved, media was replaced, and adherent cells were passaged until theyreached confluence. Media was changed every 3 days. In some experiments,cells after passage 3 were treated with either L-PBS or L-WNT3A(effective concentration=0.03 μg/mL). In these experiments, cells werecollected 24 h later and lysed using RIPA buffer (Sigma-Aldrich, St.Louis, Mo., USA). Total protein was extracted for Western analysis.Pan-actin was used as an internal control and to ensure proteinintegrity. Antibodies against WNT3A (R&D, Minneapolis, Minn., USA),non-phosphorylated β-catenin (Cell Signaling, Danvers, Mass., USA), andAxin2 (Abcam, Cambridge, Mass., USA) were used. Integrated intensity wasanalyzed by ImageJ (National institute of Health, USA version 1.47v) toquantify Western blotting results.

Sub-Renal Capsule Transplant Surgery.

In some cases, the sub-renal capsular assay (SRCA) was employed to assayits differentiation potential. Following inhalation of anesthesia by thesyngeneic host mice, a skin incision was made on the left flank directlycaudal to the rib cage. The peritoneal cavity was opened to expose thekidney. A small incision was created in the renal capsule and the BGMswere carefully placed under the capsule using soft plastic tubing. Thekidney was then returned to the peritoneal cavity, and the peritoneumand skin were closed with sutures. Buprenorphine (0.05 mg/kg) was usedfor analgesia. In cases where the BGM was harvested fromAxin2^(CreERT2); R26^(mTmG) donors, hosts were subsequently providedtamoxifen by gavage (100 μL of 10 mg/mL) beginning on day 0 for 5 days.The SRC transplants were harvested at the time points indicated. 2.6Adenovirus-mediated inhibition of Wnt signaling DKK1 and the negativecontrol Fc adenoviral constructs were described previously. Theadenoviral constructs were transfected into 293T cells. After 2 days,cells were collected, lysed, and precipitated by centrifugation. Thepurified adenovirus was aliquoted and stored at 80° C. Wnt inhibitionwas achieved by in vitro incubation of BGMs with Ad-Dkk1 and the controlAd-Fc for 2 hours, and the BGM aliquots were then transplanted intocalvarial defects.

Calvarial Critical-Size Defect Surgery.

Mice were anesthetized, and a 3-mm incision was made to expose theparietal bone. A circumferential, full-thickness defect with a 2-mmdiameter was created with the use of a micro-dissecting trephine; thedura mater was not disturbed. BGM aliquots were incubated with Ad-Dkk1and the control Ad-Fc for 2 hours. BGM aliquots were then transplantedinto the calvarial defect and the skin was closed with sutures Followingrecovery from surgery, mice received Buprenorphine for analgesia.Micro-computed tomography (Micro-CT) analyses were performed aspreviously described.

Spinal Fusion Surgery.

Lewis Rats were anesthetized using a cocktail of Ketamine 70-100 mg/kgand Xylazine 5-10 mg/kg. The lumbar region of the rats were shaved thendisinfected with Betadine soaked gauze. Prior to the skin incision, therats were injected with the analgesia buprenorphine 0.02 mg/kg SC/IP.First, bone graft material (BGM) was harvested from the iliac crest;briefly, the left iliac spine was palpated and a vertical cutaneousincision was made; the dorsal crest of the iliac spine was accessed andexposed through blunt dissection. The attached muscle and periosteumwere elevated and 0.3 g of BGM was harvested with a rongeur forceps andmorselized. BGM was then incubated with either with 100 μL L-PBS or with100 μL of [0.15 μg/mL] L-WNT3A while the transverse processes wereexposed. To expose the transverse processes, posterolateral bluntdissection was carried down and the reflected paraspinal muscles wereheld in place by retractors. The transverse processes of L4-L5 werecleaned of periosteum and decorticated with a high-speed burr. The BGMwas spread over and between the L4-L5 transverse processes. Theparaspinal muscles were closed with absorbable sutures (4-0 Vicryl) andthe skin with interrupted nonabsorbable sutures (4-0 Nylon). Thesurgical site was treated with an antibiotic ointment. 10 mg/kg Baytrilwas delivered subcutaneously. Buprenorphine (0.02 mg/kg) wasadministered after surgery for 3 days, and subsequent doses were givenas needed to control pain.

Sample Preparation, Tissue Processing, Histology.

Tissues were fixed in 4% paraformaldehyde (PFA) overnight at 4° C.Samples were decalcified in 19% EDTA for 1 day. Specimens weredehydrated through an ascending ethanol series prior to paraffinembedding. Eight-micron-thick longitudinal sections were cut andcollected on Superfrost-plus slides for histology. Safranin O, Anilineblue and Gomori staining were performed as previously described. Tissuesections were photographed using a Leica DM5000B digital imaging system(Leica Micro-systems, Wetzlar, Germany).

ALP, TRAP and TUNEL Staining.

Alkaline phosphatase (ALP) activity was detected by incubation in nitroblue tetrazolium chloride (NBT; Roche, Indianapolis, Ind., USA),5-bromo-4-chloro-3-indolyl phosphate (BCIP; Roche), and NTM buffer (100mM NaCl, 100 mM Tris pH 9.5, 5 mM MgCl). Tartrate-resistant acidphosphatase (TRAP) activity was observed using a Leukocyte acidphosphatase staining kit (Sigma, St. Louis, Mo., USA) followingmanufacturer's instructions. After developing, slides were dehydrated ina series of ethanol, cleaned in Citrisolv (Fisher Scientific), andcover-slipped with Permount mounting media (Fisher Scientific). ForTUNEL staining, sections were permeabilized using 0.1% Triton X-100(Sigma) and 0.1% sodium citrate (sigma), and incubated with TUNELreaction mixture (In Situ Cell Death Detection Kit, Roche). Sectionswere mounted with DAPI mounting medium (Vector Labs, Burlingame, Calif.,USA) and visualized under an epifluorescence microscope. Forbromodeoxyuridine (BrdU) assay, mice were given intraperitonealinjections of BrdU labeling reagent (Invitrogen, CA, USA) and euthanized4 hours post-injection. BrdU detection was carried out using BrdUStaining Kit (Invitrogen, CA, USA) following the manufacturer'sinstructions.

Immunohistochemistry.

Tissue sections were deparaffinized and rehydrated in PBS. Endogenousperoxidase activity was quenched by 3% hydrogen peroxide for 5 min, andthen washed in PBS. Slides were blocked with 5% goat serum (Vectorlaboratories) for 1 hour at room temperature. The appropriate primaryantibody was added and incubated overnight at 4° C. Samples were thenincubated with appropriate biotinylated secondary antibodies (VectorLaboratories) and advidin/biotinylated enzyme complex (VectorLaboratories) and developed by a DAB substrate kit (VectorLaboratories). Antibodies used include GFP (cell signaling) and DLK1(Millipore), Runx2 (Santa Cruz), Sox9 (Abcam) and PPAR-γ (CellSignaling).

Micro-CT Analyses and Quantification of Graft Growth.

Scanning and analyses adhered to published guidelines]. Mice wereanesthetized with 2% isoflurane and scanned with use of a multimodalpositron emission tomography computed tomography data-acquisition system(Inveon PET-CT; Siemens, Erlangen, Germany) at 40-mm resolution. Todefine the graft growth that occurred in each sample, POD2 and POD49timepoint scanning data were exported into Osirix software version 5.8(Pixmeo, Bernex, Switzerland) and registered for segmentation in thesame orientation. The new that bone formed was compared to the initialBGM volume transplanted. Differences between sets of data weredetermined by using Mann-Whitney test in XLStat software version(Addinsoft, Paris, France). A p-value<0.05 was considered statisticallysignificant.

Quantification and Statistical Analyses.

GFP, BrdU, TUNEL, DLK1, Osteocalcin and Aniline blue stainings werequantified. Photoshop CSS (Adobe, version 10.0.1) was used to determinethe number of pixels in the region of interest (ROI), at the injurysite. The magic wand tool was used to assign the area of positive pixelswithin the ROI. The ratio of pixels of positive signals to pixels of ROIwas expressed as a percentage. At least 5 sections evenly spaced acrossthe injury sites were quantified to determine the average value of eachsample. Five samples were included in each group (n=5). Results arepresented as the mean±SD. Student's t-test was used to quantifydifferences described in this article. P≦0.05 was consideredsignificant.

Results

Bone Graft Material Contains Multiple Stem/Progenitor Cell Populations.

The optimal anatomical site for harvesting autografts depends upon anumber of factors including donor site morbidity and the availability ofbone stock. We harvested BGM from three anatomical sites using amodified reamer-irrigator-aspirator (RIA) technique and noted that thefemur, iliac crest, and tibia yielded BGM with distinctly differenthistological appearances. In addition to hematopoietic cells, femur BGMcontained adipocytes, even when harvested from young animals (FIG. 7A).Iliac crest BGM was largely comprised of trabecular bone fragmentscovered in tightly adherent cells (FIG. 7B). BGM from the tibiacontained a considerable amount of fibrous stroma and small, anucleatedcells (FIG. 7C). We used quantitative RT-PCR to evaluate endogenousosteogenic gene expression and found that of the three sources, iliaccrest BGM expressed alkaline phosphatase and Osteocalcin atsignificantly higher levels (FIG. 7D). In general, it is widely believedthat the osteogenic property of an autograft is attributable tostem/progenitor cell populations and osteoblasts within the bone graftmaterial (BGM). We directly tested this hypothesis by transplanting BGMinto a sub-renal capsule (SRC) assay. The SRC provides a vascular supplyto the transplanted tissue and supports the differentiation of cellsinto multiple kinds of tissues including bone, skin, muscle, teeth,organs, and tumors. BGM was harvested from the iliac crest thentransplanted beneath the animal's kidney capsule (FIG. 7A) and allowedto develop there for 7 days. BrdU incorporation demonstrated the highmitotic activity of cells in the autologous BGM (FIG. 7B).Immunostaining for Runx2 (FIG. 7C), Sox9 (FIG. 7D) and PPARγ (FIG. 7E)demonstrated that subsets of cells in the BGM expressed gene markersassociated with osteogenic, chondrogenic and adipogenic commitment. Onday 7, a sub-population of BGM-derived cells had differentiated intobone (FIG. 7F), cartilage (FIG. 7G), and fat (FIG. 7H). Together, thesedata demonstrated that the BGM contains stem/progenitor cells capable ofdifferentiating into all three lineages.

Wnt Signaling in Bone Graft Material Declines with Age.

Wnts are among the best studied of the molecular signals that induceosteogenic differentiation. Using Axin2^(CreERT2); R26m^(TmG) reportermice we induced recombination (see Methods) then identified GFP^(+ve)pre-osteoblasts in the periosteum (FIG. 8A) and the endosteum (FIG. 8B).The frequency of GFP^(+ve) cells in the endosteum was 0.1% (FIG. 8C).GFP^(+ve) cells were also identified in freshly harvested BGM (FIG. 8D).Thus, a subset of cells in the heterogeneous BGM is Wnt responsive. Wecompared the Wnt responsive status of BGM from young (<3 month old) andaged (>10 month old) mice. Quantitative absolute RT-PCR demonstratedthat expression of the Wnt target genes Axin2 and Lef1 was almosttwo-fold lower in BGM harvested from aged mice (BGM^(aged)) v. youngmice (BGM^(young); FIG. 8F). Western analysis confirmed that Wnt3a,phosphorylated beta catenin, and Axin2 expression were all significantlylower in BGM^(aged) compared to BGM^(young) (FIG. 8G). Thus, theendogenous Wnt responsive status of BGM deteriorates with age.

Osteogenic Differentiation Capacity Also Declines with Age.

In humans, the rate of bone healing declines with age. We found asimilar age-related decline in the osteogenic capacity of BGM. Freshlyharvested BGM^(aged) showed significantly lower expression levels of theosteogenic genes Alkaline phosphatase, Osterix, and Osteocalcin comparedto freshly harvested BGM^(young) (FIG. 9A). To test whether thereduction in osteogenic gene expression affected the osteogenic capacityof the BGM we returned to the SRC assay. Performing an autograft in amouse, however, is excessively traumatic. To mimic an autograft, we usedsyngeneic donors and hosts. Because syngeneic animals are so closelyrelated, their tissues are immunologically compatible andtransplantation of tissues does not provoke an immune response.ACTB-eGFP mice served as the donors and BGM was readily identifiable inthe SRC by its GFP fluorescence (FIG. 9B,C).

Seven days after transplantation, BGM was harvested and analyzed forevidence of bone formation. Aniline blue stained osteoid matrix wasevident in BGM^(young) (FIG. 9D) but absent in BGM^(aged) (FIG. 9E;quantified in F). The osteoid matrix in BGM^(young) was undergoingmineralization as shown by ALP staining (FIG. 9G) whereas BGM^(aged)showed no ALP staining (FIG. 9H; quantified in I). We wondered ifengraftment efficiency between BGM^(aged) and BGM^(young) could accountfor the differences in osteogenic differentiation but GFP immunostainingdemonstrated that in both BGM^(young) (FIG. 9J) and BGM^(aged) (FIG. 9K)there were a similar number of surviving donor cells (quantified in FIG.9L). Together these data indicate that osteogenic gene expression andosteogenic capacity of BGM declines with age.

Wnt Signaling is Necessary for the Osteogenic Capacity of BGM.

Endogenous Wnt responsiveness, and the osteogenic capacity of BGM,diminishes with age. To test whether reduced Wnt signaling contributedto this age-related decline in osteogenic potential, we blockedendogenous Wnt signaling in BGM. Others and we have used over-expressionof the Wnt inhibitor, Dkk1 to transiently abolish Wnt signaling in vivo.We delivered either Ad-Dkk1 or Ad-Fc (control) to the bone marrow cavityof young mice then harvested BGM^(young) 24 h later and transplanted thealiquots into critical size (non-healing) skeletal defects. Seven dayslater, when control BGM^(young) was strongly positive for ALP activity(FIG. 10A), Ad-Dkk1 treated BGM^(young) showed minimal activity (FIG.10B). Instead, Ad-Dkk1 treated BGM^(young) showed widespread expressionof the adipogenic proteins PPAR-□□ (FIG. 10C,D) and Dlk1 (FIG. 10E,F).Bone formation was repressed by Ad-Dkk1 treatment, as shown by micro-CT(FIG. 10G,H; quantified in I) and histomorphometric analyses of the BGM(FIG. 10J,K; quantified in L). Thus, the osteogenic capacity of BGMrelies upon an endogenous Wnt signal.

Augmenting the Endogenous Wnt Signal in BGM^(aged) Restores itsOsteogenic Capacity.

Endogenous Wnt signaling is necessary for BGM to exhibits its osteogeniccapacity (FIG. 10J-L). We next tested whether a Wnt stimulus wassufficient to enhance BGM efficacy. BGM^(aged) was harvested, treatedwith L-WNT3A or liposomal PBS (L-PBS) then incubated at 37° C. (FIG.11A). Absolute qRT-PCR analyses revealed a small but significantelevation in Axin2 expression (FIG. 11B). Lef1 was modestly elevated inresponse to L-WNT3A (FIG. 11B). Western analyses indicated that bothbeta catenin and Axin2 proteins were elevated in response to L-WNT3A(FIG. 11C). Mitotic activity in BGM was increased by L-WNT3A treatment.On post-transplant day 4, cell proliferation was significantly increasedin L-WNT3A treated BGM^(aged) compared to LPBS-treated BGM^(aged) (FIG.11D,E; quantified in F). The effect on cell cycling was transient: bypost-transplant 7, BrdU incorporation was equivalent between the L-PBSand L-WNT3A samples (FIG. 11G,H; quantified in I). Cell differentiationin BGM^(aged) was evaluated. Expression of the adipogenic protein Dlk1was lower (FIG. 11J,K; quantified in L) and expression of the osteogenicprotein Osteocalcin was higher in L-WNT3A treated BGM^(aged) (FIG.11M,N; quantified in O). New bone formation was found only in L-WNT3Atreated BGM^(aged) (FIG. 11P,Q; quantified in R). Treatment with LWNT3Adid not affect engraftment efficiency but analyses of programmed celldeath demonstrated that L-WNT3A treated BGM^(aged) had significantlyfewer TUNEL^(+ve) cells than L-PBS treated samples. Reduced apoptosiswas also observed at post-transplant day 7. New bone formation serves asa stimulus for osteoclast-mediated bone remodeling and we observed TRAPactivity around the newly formed osteoid matrix only in L-WNT3A treatedBGM^(aged) samples. Thus we conclude that L-WNT3A is sufficient toenhance the osteogenic capacity of BGM.

L-WNT3A Activates Stem Cells in BGMaged and Improves Bone Generation ina Spinal Fusion Model.

We sought to identify the population of cells in BGM responsible for theL-WNT3A mediated surge in osteogenic capacity. We isolated three stemcell populations from BGM using standard procedures then evaluated theirWnt responsiveness using LPBS (as control) or L-WNT3A. In previousexperiments we determined a dose of L-WNT3A that reliably activatedAxin2 expression in stem cell populations. Time-course analyses revealedthe response of stem cells to L-WNT3A treatment: within 15 h of L-WNT3Aexposure a 4-fold activation in Axin2 was observed, and maximal Axin2activation was achieved at 36 h (FIG. 12A). The effect was transient, asshown by diminished Axin2 expression in stem cells at the 60 h timepoint(FIG. 12A).

We used a second stem cell population isolated from BGM to verify thatstem cells respond to L-WNT3A (FIG. 12B). The activated state of the BGMwas shown by qRT-PCR. BGM^(aged) was harvested, treated with L-WNT3A orL-PBS then analyzed using Axin2 and Lef1 expression for its Wntresponsive status (FIG. 12C). Within 12 h a significant elevation inLef1 was detectable; within 24 h, both Axin2 and Lef1 were significantlyelevated (FIG. 12C). These analyses confirm a MINT-mediated activationstate of BGM; hereafter we refer to this material as BGM^(ACT). Our nextexperiments tested the therapeutic potential of BGM^(ACT) in a ratspinal fusion model. The transverse processes of the fourth and fifthlumbar (e.g., L4-5) vertebrae were decorticated (FIG. 12D) and duringthis procedure, autologous BGM^(aged) from the iliac crest was harvestedand treated with L-WNT3A (or L-PBS) for 1 h. The resulting material,BGM^(ACT) (or BGM^(aged)) was then transplanted onto and between theL4-5 processes (FIG. 12E). On post-operation day 2 the volume of the BGMwas evaluated by micro-CT; these analyses verified that BGM^(aged) (FIG.12F) and BGMACT (FIG. 12) contained comparable amounts of mineralizedtissue at the outset. The volume of new bone formation was re-evaluatedon post-operation day 49. Three dimensional reconstructions of themicro-CT data demonstrated poor bone regeneration in sites treated withBGM^(aged) (grey; FIG. 12G), in agreement with similar data from elderlypatients undergoing spine fusion. In contrast, sites treated withBGM^(ACT) showed evidence of robust bone formation and fusion of thetransverse processes (blue; FIG. 12H). The volume of new bone betweenthe transverse processes was quantified; compared to BGM^(aged),BGM^(ACT) gave rise to significantly more mineralized matrix (FIG. 12I).Thus, L-WNT3A treatment improves the osteogenic capacity of autograftsfrom aged animals.

Almost half a million bone grafting procedures are performed annually,making autografts the second most commonly transplanted tissue in theUnited States (AATB Annual Survey,). Autografts have significantadvantages over allogeneic grafts and synthetic bone substitutes, butthey are contraindicated in the elderly and in patients with underlyingbone or metabolic diseases. Here, we directed our efforts towardsunderstanding the factors important for autograft efficacy, and towardsvalidating a method that improves autograft efficacy. Four major factorsinfluence the osteogenic capacity of an autograft: first, the site fromwhich the autograft is harvested; second, how the autograft is handledafter harvesting; third, the growth factor constituency of theautograft; and fourth, the activation state of stem cells in theautograft. Here, we provide evidence that WNT signals can influencethree of these four critical variables.

Optimizing autograft harvesting and handling. The osteogenic capacity,and thus the efficacy of an autograft, is influenced by the site andmethod of harvest. Most non-vascularized autografts are harvested fromthe iliac crest but with a reamer/irrigator/aspirator (RIA) approach,the femoral and tibial medullary cavities can also be accessed. Theosteogenic capacity of autografts collected via RIA and conventionalharvest is equivalent. Using a simulated RIA approach we observed adistinct difference in the cellular constituency of BGM harvested fromthe iliac crest, the femur, and the tibia. Further, iliac crest BGM hada significantly higher level of anabolic osteogenic gene expressioncompared to BGM from the femur or tibia (FIG. 7). All of these BGMaliquots, however, exhibited bone-forming potential in the sub-renalcapsule (SRC) assay (FIG. 7). The efficacy of an autograft can also becompromised by inappropriate handling of the material, prior totransplantation back into the patient. For example, even when freshlyharvested BGM is maintained at room temperature there is significantcellular apoptosis. Treating BGM with L-WNT3A significantly improvescell survival: for example, compared to BGM alone, BGM^(ACT) exhibits50% fewer TUNEL-positive cells. Mitotic activity is also significantlyhigher in BGM^(ACT) v. BGM treated with L-PBS (FIG. 11D-F). Together,this increase in cell proliferation and a concomitant reduction in celldeath translate into increased BGM viability and thus improved autograftefficacy.

Optimizing growth factor activity in autografts. The efficacy of anautograft also appears to depend upon the presence of growth factors inthe material. In freshly harvested autografts, a wide variety of growthfactors have been identified including transforming growth factor beta,bone morphogenetic proteins, vascular endothelial growth factor, andplatelet-derived growth factor. In demineralized freeze-driedallografts, however, these factors are either lacking or present inminimally measurable quantities. To our knowledge there are no studiesreporting the level of endogenous WNT signaling in autografts orallografts. There are, however, a number of studies clearlydemonstrating that serum levels of Wnt inhibitors are elevated in theelderly, which presumably decreases Wnt signaling activity. Theseclinical findings are in agreement with our data showing that endogenousWnt activity in BGM declines with age (FIG. 8). Consequently, anapproach that elevates WNT responsiveness restores osteogenic capacityto autografts. We demonstrated that treatment with L-WNT3A activates Wntsignaling in the BGM, which correlates with robust osteogenesis in theSRC (FIG. 11) and in a spinal fusion model (FIG. 12).

Activating autograft stem cells with L-WNT3A. At least some of theefficacy of an autograft can be traced back to stem/stromal cells withinthe material. In the marrow cavity, osteogenic/skeletal stem cells areadhered to or embedded on the endosteal surface. As a consequence,harvesting methods that rely on aspiration alone typically fail tocollect these adherent stem cell populations. In fact, current estimatesplace the number of stem cells in bone marrow aspirates as low as1/50,000 nucleated cells; in elderly patients the number drops to only1/1,000,000 nucleated cells. RIA harvesting intentionally removes theendosteal surface and therefore is more likely to contain the osteogenicstem cell populations. We used a modified RIA approach that removes thesurface of the endosteum where Wnt responsive cells reside (FIG. 8) thendemonstrated that BGM collected in this manner contains stem/progenitorcell populations and is robustly osteogenic (FIG. 7), specificallybecause of the endogenous Wnt signal (FIG. 10).

Autografts continue to represent the classic exemplar for bonyreconstruction; there still remains, however, considerable room forimprovement in autograft efficacy. Data shown here demonstrate that exvivo exposure to L-WNT3A improves cell viability and activates stem cellpopulations in freshly harvested autografts, which culminates inincreased osteogenic activity. These data have direct clinicalapplication, especially for autografts from at-risk patient populationswhose inherent bone forming capacity is reduced by illness, disease, oraging.

1-58. (canceled)
 59. An effective dose of an isolated enhanced mammalianbone graft material comprising enhanced cells wherein the enhanced cellshave an increased level of expression for one or more biomarkerscomprising Axin2, Tcf4, or Lef1 after exposure ex vivo with a liposomecomprising a Wnt protein, wherein the increased level of expression forthe one or more biomarkers is relative to equivalent cells from isolatedmammalian bone graft material unexposed to a liposome comprising a Wntprotein.
 60. The effective dose of claim 59, wherein the enhanced cellsfurther have an increased level of expression for one or more biomarkerscomprising Runx2, Osterix, Osteocalcin, or alkaline phosphatase.
 61. Theeffective dose of claim 59, wherein the increased level of expression isincreased level of gene expression.
 62. The effective dose of claim 59,wherein the increased level of expression is increased level of proteinexpression.
 63. The effective dose of claim 59, wherein the increasedlevel of expression is up to 9× relative to the equivalent cells fromisolated mammalian bone graft material.
 64. The effective dose of claim59, wherein the Wnt protein is Wnt3a protein.
 65. The effective dose ofclaim 59, wherein the enhanced cells comprise bone marrow stem cells orbone marrow progenitor cells.
 66. The effective dose of claim 59,wherein the enhanced cells comprise mesenchymal stem cells orosteocytes.
 67. The effective dose of claim 59, wherein the isolatedenhanced mammalian bone graft material comprises enhanced cells withreduced apoptosis after exposure ex vivo with a liposome comprising aWnt protein, wherein the reduced apoptosis is relative to equivalentcells from isolated mammalian bone graft material unexposed to aliposome comprising a Wnt protein.
 68. The effective dose of claim 67,wherein the reduced apoptosis in the enhanced cells is measured asreduced caspase activity relative to equivalent cells from isolatedmammalian bone graft material unexposed to a liposome comprising a Wntprotein.
 69. The effective dose of claim 59, wherein the isolatedenhanced mammalian bone graft material comprises enhanced cells withincreased cell proliferation after exposure ex vivo with a liposomecomprising a Wnt protein, wherein the increased cell proliferation isrelative to equivalent cells from isolated mammalian bone graft materialunexposed to a liposome comprising a Wnt protein.
 70. The effective doseof claim 69, wherein the increased cell proliferation in the enhancedcells is measured as increased mitotic activity relative to equivalentcells from isolated mammalian bone graft material unexposed to aliposome comprising a Wnt protein.
 71. The effective dose of claim 59,wherein the isolated enhanced mammalian bone graft material is isolatedenhanced human bone graft material.
 72. The effective dose of claim 59,wherein the isolated enhanced human bone graft material is obtained froma human subject at least 50 years of age or older.
 73. An ex vivo methodof enhancing cell survival in a bone graft in a subject in need thereof,comprising: a) incubating a sample comprising isolated mammalian bonegraft material comprising cells ex-vivo with a liposome comprising a Wntprotein to produce enhanced cells comprising an increased level ofexpression for one or more biomarkers comprising Axin2, Tcf4, or Lef1;and b) transplanting the enhanced cells into a target site on bone. 74.The ex vivo method of claim 73, wherein the enhanced cells further havean increased level of expression for one or more biomarkers comprisingRunx2, Osterix, Osteocalcin, or alkaline phosphatase.
 75. The ex vivomethod of claim 73, wherein the incubating temperature is at roomtemperature or at about 37° C.
 76. The ex vivo method of claim 73,wherein the incubating time is from about 1 hour to about 36 hours. 77.The ex vivo method of claim 73, wherein the isolated mammalian bonegraft material further comprises enhanced osteogenic capacity comparedto unexposed mammalian bone graft material.
 78. The ex vivo method ofclaim 77, wherein the enhanced osteogenic capacity comprises increasedvolume of bone growth of about 1.5-fold, about 2-fold, about 3-fold, ormore compared to the volume of bone growth of unexposed mammalian bonemarrow cells.
 79. The ex vivo method of claim 73, wherein the increasedlevel of expression of the enhanced cells is relative to equivalentcells from isolated mammalian bone graft material without incubationwith a liposome comprising a Wnt protein.
 80. The ex vivo method ofclaim 73, wherein the increased level of expression is increased levelof gene expression.
 81. The ex vivo method of claim 73, wherein theincreased level of expression is increased level of protein expression.82. The ex vivo method of claim 73, wherein the increased level ofexpression is up to 9× relative to the equivalent cells from isolatedmammalian bone graft material.
 83. The ex vivo method of claim 73,wherein the enhanced cells comprise bone marrow stem cells or bonemarrow progenitor cells.
 84. The ex vivo method of claim 73, wherein theenhanced cells comprise mesenchymal stem cells or osteocytes.
 85. The exvivo method of claim 73, wherein the isolated mammalian bone graftmaterial is isolated human bone graft material.
 86. The ex vivo methodof claim 73, wherein the isolated mammalian bone graft material isobtained from a human subject at least 50 years of age or older.
 87. Anex vivo method of enhancing cell survival in a bone graft in a subjectin need thereof, comprising: a) isolating a bone graft sample comprisingcells from a human subject, b) incubating the bone graft sample ex-vivowith a liposome comprising a Wnt3a protein for a sufficient time toproduce enhanced cells comprising an increased level of expression forone or more biomarkers comprising Axin2, Tcf4, or Lef1 relative toequivalent cells from bone graft material not incubated with a liposomecomprising a Wnt3a protein; and c) transplanting the incubated enhancedcells into a target site on bone.
 88. The ex vivo method of claim 87,wherein the enhanced cells further have an increased level of expressionfor one or more biomarkers comprising Runx2, Osterix, Osteocalcin, oralkaline phosphatase.